Briefly, a sequential dual immunostaining protocol was used for the simultaneous visualization of α-synuclein and tau. The colorimetric detection of α-synuclein was carried out firstly using a Bond Polymer Refine DAB detection kit (Leica) resulting in a brown precipitate. Next, the Bond Polymer Refine Red detection kit (Leica) was used to visualize the expression of tau protein by means of a red-colored precipitate.
Bond polymer refine dab detection kit
Manufactured by Leica
Sourced in United States, Germany
The BOND Polymer Refine DAB Detection kit is a laboratory equipment product designed for immunohistochemistry (IHC) and immunocytochemistry (ICC) applications. It provides a sensitive and reliable method for detecting target antigens in tissue sections or cell samples. The kit utilizes a polymer-based detection system and a 3,3'-Diaminobenzidine (DAB) chromogen, enabling the visualization of target proteins.
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Lab products found in correlation
Bond rx, by Leica (3 mentions)
Bond 3, by Leica (2 mentions)
Bond polymer refine red detection kit, by Leica (1 mentions)
Cytoseal 60, by Thermo Fisher Scientific (1 mentions)
Ab152, by Novus Biologicals (1 mentions)
Ventana discovery ultra, by Roche (1 mentions)
Bond primary antibody diluent, by Leica (1 mentions)
Pannoramic p250, by 3DHISTECH (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Image pro premier software, by Media Cybernetics (1 mentions)
Bond max autostainer, by Leica (1 mentions)
Ab 217787, by Abcam (1 mentions)
Define mx aq, by Leica (1 mentions)
Hematoxylin 560 mx, by Leica (1 mentions)
Eosin phloxine 515, by Leica (1 mentions)
10 protocols using bond polymer refine dab detection kit
1
Dual Immunostaining of α-Synuclein and Tau
2
Immunohistochemical Analysis of Bone Marrow
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Femurs (one each from two mice per group for 8wk HU and both femurs for one mouse per group for HUR) were carefully excised and fixed in 10% neutral buffered formalin, decalcified with Immunocal Decalcifier (Statlab 1414-1), processed (Sakura Tissue-Tek VIP5), embedded in paraffin, sectioned at 4 µm, and mounted on plus slides. The slides were stained with Tyrosine Hydroxylase antibody (EMD Millipore, AB152 1 : 500) or Leptin/OB (Novus Biologicals MAB498, 1 : 4000) and the BOND Polymer Refine DAB Detection kit (Leica DS9800) on a Leica BOND-MAX immunostaining platform, dehydrated in graded alcohols, cleared in xylene, and cover slipped with Cytoseal 60 (Thermo Scientific 8310). For each stain, four sections per femur were analyzed. The BM of each IHC section was randomly photographed at 100 × 3 times in each of three regions: proximal (in or near the femoral head), medial (within the shaft of the bone), and distal (in or near the epicondyles). Micrographs were scored using the IHC Profiler108 (link) plugin for ImageJ, which allows for non-biased, automated scoring of histological slides. In this method, each DAB-stained pixel is categorized into one of four pre-set pixel intensity bins (High Positive, Positive, Low Positive, Negative) and yields the percent contribution of each bin to the image.
3
In Situ RNA Amplification for MALAT1
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The in situ RNA amplification and labeling process were performed on the automated ISH platform Ventana Discovery ULTRA (Ventana Medical System Inc.) or the Bond RX (Leica Biosystems) for the probe detection RNAscope 2.5 VS Reagent Kit-RED and Bond Polymer Refine DAB Detection Kit, respectively. ISH probe for MALAT1 and RNAscope VS or LS Assay reagents were obtained from Advanced Cell Diagnostics (ACD) (Newark, CA) and used according to the manufacturer’s instructions. Stained slides were analyzed under a standard bright-field microscope.
4
Automated IHC Staining for RORC(t) Expression
5
Smarcd3 Immunohistochemistry Protocol for TMAs
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TMAs were sectioned to 2.5 µm thickness. IHC staining was performed on a Leica BOND RX automated immunostainer using BOND primary antibody diluent and BOND Polymer Refine DAB Detection kit per manufacturer’s instructions (Leica Biosystems). Pre-treatment was performed using citrate buffer at 100 °C for 30 min, and tissue was stained using rabbit anti-human Smarcd3 antibody (Aviva Systems Biology). Slides were scanned using a Pannoramic P250 digital slide scanner (3DHistech). Smarcd3 staining of TMA spots was analyzed in an independent and randomized manner by two board-certified surgical pathologists (C.M.S and M.W.) using Scorenado, a custom-made online digital TMA analysis tool. Interpretation of staining results was in accordance with the “reporting recommendations for tumor marker prognostic studies” (REMARK) guidelines. Equivocal and discordant cases were reviewed by a third board-certified surgical pathologist (E.K.) to reach a consensus. Smarcd3 staining in tumor cells was classified microscopically as negative (absence of any staining), vs. positive (any positive staining in tumor cells). Spots/patients with no interpretable tissue (less than 10 intact, unequivocally identifiable tumor cells) or other artifacts were excluded.
6
Immunohistochemical Analysis of CK19 and 5hmC
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In brief, tissues were sectioned (5 μm), de-paraffinized, and re-hydrated using ethanol and xylene. Slides were treated for 10 min with 3% H2O2 within the PBS to weaken the activity of endogenous peroxidase, followed by overnight incubation using rabbit monoclonal anti-CK19 antibody (1:1500, ab15580) and anti-5hmC (1:800, ab214728) antibody according to specific Leica Bond protocols IHC-F. Additionally, this study employed a Leica Bond Polymer Refine DAB detection kit for instruction. Two hepatologists evaluated overall IHC slides; corresponding scoring approaches were carried out as introduced by the former report [9 (link)].
According to CK19 or 5hmC positive staining, if more than 80% cells displayed nuclear positivity, the cells were scored positive and negative otherwise.
According to CK19 or 5hmC positive staining, if more than 80% cells displayed nuclear positivity, the cells were scored positive and negative otherwise.
7
Quantitative Tissue Analysis by H&E and IHC
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Haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed for tissue processed after tissue maintenance in the MF device as well as those obtained fresh from the patient. Tissues were initially placed in 1.5 ml of 4% paraformaldehyde and processed for paraffin embedding. Wax-embedded tissues were cut (5 μm) and stained with H&E. IHC was performed using the Leica Bond III automated immunostaining platform (Leica) in conjunction with the Leica Bond Polymer Refine DAB detection kit (Leica), as per the manufacturer's guidelines. Ki67 and Caspase 3 proteins were targeted with the respective antibodies, MIB1 and ASP175. All H&E and IHC slides were viewed using an Eclipse 80i microscope (Nikon), and digital images were acquired using an Infinity 3 Microscope Camera (Roper Technologies) along with Image Pro Premier Software (Media Cybernetics). Digital images of the IHC slides were analysed using ImmunoRatio, a free Web-based application for automated image analysis of stained tissue sections [[21] , [22] (link), [23] ]. Proliferation and apoptotic indices were calculated as the number of positively stained cells divided by the total number of cells, multiplied by 100. For each slide, this was performed on 2–3 randomly chosen high-power field images that contained positively stained cells.
8
Immunohistochemical Analysis of H3K27me3 Expression
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IHC analyses were performed as reported [9 (link)]. In brief, tissue sections were cut at 5 μm, followed by deparaffinization and rehydration using xylene and ethanol. Then, the slides were incubated in 3% hydrogen peroxide for 10 min in phosphate-buffered saline to block endogenous peroxidase activity. Slides were incubated overnight with rabbit monoclonal anti-H3K27me3 antibody (C36B11, Cell Signaling, Danvers, MA, USA) at a concentration of 1:150 using the standard Leica Bond protocol IHC-F. The Leica Bond Polymer Refine DAB detection kit was used according to the manufacturer’s instructions. All IHC slides were evaluated by two independent neuropathologists; the scoring methods were performed as report described [9 (link)]. H3K27me3 positive staining was defined as scored positive when more than 80% cells had nuclear positivity and scored negative when they did not.
9
Immunohistochemical Analysis of RNF43 and LRP1B
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Immunohistochemical staining was performed on tissue sections of the primary tumor using the Bondmax Autostainer (Leica Biosystems, Germany) and polyclonal antibodies directed against RNF43 (ab 217787, Abcam, Cambridge, UK) and LRP1B (HPA069094, Sigma-Aldrich, St. Louis, MO, USA) in 1:100 dilutions27 ,28 . All tissue sections were pretreated for 20 min with ER1-antigen retrieval solution (Leica Biosystems, Germany). For visualization the Bond Polymer Refine Detection Kit DAB (Leica Biosystems, Germany) was used. Counterstaining was performed with hematoxylin.
10
Immunohistochemical Analysis of HNSCC
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The hematoxylin and eosin (H&E) and CD8 IHC staining on HNSCC specimens was performed at UPMC Developmental Laboratory. Slides were cut at 4μm then baked for one hour at 60 degrees Celsius. The slides were cooled to room temperature then deparaffinized and hydrated in diH20. Slides were stained using Hematoxylin 560 MX (3801576, Eosin Phloxine 515 (3801606), Define MX-aq (3803598) and Blue Buffer 8 (3802918) (all catalog numbers from Leica Biosystems). The predilute CD8 (Leica Biosystems, PA0183) was stained on the Bond III instrument (Leica Biosystems) for 8 min. Prior to antibody placement, the antigen retrieval used was the ER2 (pH 8.9–9.1, Leica Biosystems, AR9640) for 20 minutes at 95 deg. Followed by the Bond Polymer Refine Detection Kit-DAB (Leica Biosystems, DS9800).
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